Method for the production of (r)-and (s)-8-chloro-6-hydroxy-octanic acid alkyl esters by enzymatic reduction

ABSTRACT

The invention relates to a method for the production of (R)- or (S)-8-chloro-6-hydroxyoctanoic acid alkyl esters of the general formula (R)-II or (S)-II  
                 
 
in which R has the meaning C 1-4 -alkyl, from 8-chloro-6-oxooctanoic acid alkyl esters of the general formula I  
                 
 
in which R has the above meaning, by enzymatic reduction using alcohol dehydrogenases, such as  Lactobacillus brevis  or  Thermoanaerobium brokii , in the presence of cofactor regeneration systems. 
The resulting (R)- and (S)-8-chloro-6-hydroxyoctanoic acid esters can be converted in a known manner into (R)-α-lipoic acid and (S)-α-lipoic acid, respectively.

The invention relates to a method for the production of (R)- and(S)-8-chloro-6-hydroxyoctanoic acid alkyl esters of the formula I byenzymatic reduction of a suitable prochiral keto compound.

The synthesis of α-lipoic acid on the industrial scale starts from8-chloro-6-oxooctanoic acid alkyl esters, which are converted by NaBH₄reduction into B-chloro-6-hydroxyoctanoic acid alkyl esters (reference:Kleenann and Engel, Pharmaceutical Substances, 3rd Ed., Thieme, 1999, p.1860). A subsequent three-stage synthetic sequence results in theracemic α-lipoic acid in a high overall yield. Besides the synthesis ofraceric α-lipoic acid, also of great importance is the synthesis of thepure enantiomers for specific pharmaceutical use (concerning this, see,for example EP 04 27 247). It is appropriate to carry out the synthesisof the pure enantiomers in analogy to the established economic methodfor synthesizing the racenic active ingredient. Accordingly, variousmethods have been developed to prepare the enantiopure intermediates,see, inter alia, DE-A-19533882. Enantio-selective reductions ofprochiral ketone compounds to chiral secondary alcohols, which lead asintermediates in alternative synthetic routes to enantiopure α-lipoicacids, are to be found in DE-A-197 09 069. All the methods involvemultistage synthetic sequences, some of which are associated withcomplicated purification operations or else are based on racemateresolution (DE 4137773), leading to the maximum yield of an enantiomerbeing 50%—without recycling by racemization or inversion. The lowoverall yield of these methods make them appear economicallyunrewarding. None of the described methods is based on a direct,one-stage preparation of an enantiopure intermediate of the establishedeconomic method for synthesizing racemic α-lipoic acid.

The object of the invention was thus to indicate a method for theproduction of certain intermediates for producing (R)- and (S)-α-lipoicacid and of (R)- and (s)-α-lipoic acid itself, which makes it possibleto produce these compounds and the intermediates with high yield andhigh enantiopurity.

This object is achieved by enzymatic reduction of 8-chloro-6-oxooctanoicacid alkyl esters using alcohol dehydrogenases or carbonyl reductases.This conversion results in either the (R) or(S)-8-chloro-6-hydroxyoctanoic acid alkyl ester of the formula (R)-II or(S)-II indicated in the claim.

The invention derives from the realization that the employed initialester can be reduced in a simple manner and very effectively using knownalcohol dehydrogenases or carbonyl reductases.

There are certain indications in the literature that dehydrogenasesmight be suitable for synthesizing chiral compounds (see, inter alia,Kragl and Kula, in. Stereoselective Biotransformations, editor R. Patel,Marcel Dekker, 2000, pages 839-866). However, the general statements inthis and similar references cannot be applied to complex startingcompounds. The worry for the skilled worker in this connection isordinarily side reactions and reduced enantio-selectivity. Thus, onlyone example of the biocatalytic reduction of the chloroethyl ketonecould be found in the literature (Mele at al., J. Org. Chem. 1991, 56,6019). In this case, a chloroethyl aryl ketone is reduced to the chiralsecondary alcohol by whole-cell biotransformation (Saccharomycescerevisiae). The reduction proceeds with neither high chemoselectivitynor high enantioselectivity. In the reduction of chloroethyl ketoneswith biocatalysts which, as in the case of compounds of the formula I,have a second bulky substituent, it is not possible to predict, owing tothe large spatial demands of the substituents, whether a particularbiocatalyst will accept such a compound as substrate.

J. Org. Chem. 66, 8682-84 (2001) reveals that an8-chloro-3-hydroxyoxtanoic acid alkyl ester can be obtained by reductionfrom the corresponding ketone with purified carbonyl reductase and wholecells. EP 0 939 132 A1 discloses an enzymatic reduction of4-halo-3-ketobutyric acid esters. J. Org. Chem. 63, 1102-08 (1998)describes the reduction of 3-chloro-4-ketooctanoic acid ethyl esters. EP0 487 986 A2 discloses obtaining (3S)-3-hydroxyoctanedioic acid diestersfor preparing lipoic acid by reduction with baker's yeast.

Surprisingly, various alcohol dehydrogenases and carbonyl reductasesshow high acceptance of compounds of the formula I as substrate(analytical assay), and it has been possible to confirm this inpreparative conversions.

The invention is indicated more precisely in claim 1 and furtherdependent claims. In the conversion of the invention, generally knowncofactors are employed, such as, for example, NAD(H), NADP(H), FADH₂.NAD(H) or NADP(H) is preferably used.

The configuration of the resulting 8-chloro-6-hydroxyoctanoic acid alkylesters is determined by the enzyme employed. Thus, reduction of8-chloro-6-oxooctanoic acid alkyl esters using alcohol dehydrogenasefrom Thermoanaeorubium brokii results in enantiopure(R)-8-chloro-6-hydroxyoctanoic acid alkyl esters. In the case ofreduction using Lactobacillus brevis alcohol dehydrogenase, there isenantioselective formation of (S)-8-chloro-6-hydroxyoctanoic acid alkylesters (ee>65%). Enzymes which show an activity with the compounds ofthe formula I as substrate are listed in the table below.

The enzymes are commercially available.

The starting compounds for preparing the intermediates, the prochiral8-chloro-6-oxooctanoic acid alkyl esters, can be obtained in a known way(L. J. Reed et al., J. Am. Chem. Soc. 1955, 774, 416).

-   Y-ADH yeast alcohol dehydrogenase-   HL-ADH horse liver ADH-   READH Rhodococcus erythropolis ADH-   CPCR Candida parapsilosiscarbonyl reductase-   CBADH Candida boidinii ADH-   LKADH Lactobacillus kefir ADH-   LEADH Lactobacillus brevis ADH-   TEADH Thermoanaerobium brokii ADH-   TEA triethanolamine-   Tris trishydroxymethylaminomethane-   Kpi mixture of monopotassium phosphate and dipotassium phosphate

DTT dithiothreitol Cosubstrate/ Activity/ Enzyme Cofactor BufferCoenzyme (conversion) Standard Y-ADH NAD(H) 100 mM TEA, 20% 2-butanonepH 7.0 HL-ADH NAD(H) 100 mM TEA, <1% methyl 1 mM MgCl₂ aceto- pH 7.0acetate READH NAD(H) 100 mM  2% methyl glycyl- aceto- glycine, pHacetate 6.5 CBADH NAD(H) 50 mM Kpi, (30° C.) 60% acetone pH 6.5 CPCRNAD(H) 100 mM TEA, (HCO₂Na, 60% methyl pH 7.8 FDH) aceto- acetate LKADHNADP(H) 50 mM Kpi, iso-propanol 10% aceto- 0.1 mM MgCl₂, 200 mM (36%)phenone pH 7.0 LBADH NADP(H) 50 mM Kpi, iso-propanol  6% methyl 1 mMMgCl₂, 200 mM (>25%) aceto- pH 6.5 acetate TBADH NADP(H) 50 mM Tris,HCO₂Na,  6% 2-butanone 1 mM DTT, FDH (>85%) pH 7.8 (37° C.)

For preparative conversions it proves advantageous to include a cofactorregeneration system in the enzymatic biotransformation. Cofactorregeneration systems which prove to be particularly advantageous arethose which shift the equilibrium of the main reaction. Thus, forexample in the case of reductions with Lactobacillus brevis alcoholdehydrogenase, a substrate-coupled co-factor regeneration in thepresence of an excess of a secondary alcohol (e.g. 2-propanol) ispossible and advantageous. Alternatively, enzyme-coupled cofactorregeneration systems (e.g. formate dehydrogenase (FDH)/formate) can beemployed, and can be utilized for the reduction of NAD(P) to NAD(P)H.The CO₂ resulting from the oxidation of the cosubstrate sodium formateescapes as gas and is thus removed from the equilibrium. Both NAD- andNADP-dependent formate dehydrogenases are described in the literatureand commercially available.

The absolute configuration of the optical isomers of8-chloro-6-hydroxyoctanoic acid alkyl esters of the formula I wasdetermined by comparison with the signs of the specific opticalrotations with literature data (Gewald et al., DE 195 33 881). Inaddition, the relative contents of the optical isomers of the8-chloro-6-hydroxyoctanoic acid alkyl esters of the formula I was foundby GC on columns with chiral phase with a limit of detection of <0.5%.

The present invention makes it possible to obtain the 20 (R)- and(S)-8-chloro-6-hydroxyoctanoic acid alkyl esters of the formula I in asimple and economic manner in high chemical and optical yield(theoretically 100% chemical and optical yield) in a one-stage method.

The invention also relates to the use of the enantiopure octanoic acidalkyl esters obtained in the method of the invention for producing (R)-or (S)-α-lipoic acid in a manner know per se. In the known methods, thechlorohydroxyoctanoic acid alkyl esters are normally converted into thecorresponding dichlorooctanoic acid alkyl esters. The known lipoic acidstructure is then obtained in a further reaction step by introducingsulfur.

As an example of the enantioselective production of an8-chloro-6-hydroxyoctanoic acid alkyl ester in accordance with thepresent invention, the following scheme shows the production ofenantiopure (R)-8-chloro-6-hydroxyoctanoic acid methyl ester.

(S)-8-Chloro-6-hydroxyoctanoic acid alkyl esters are obtainable in ananalogous manner by employing as biocatalyst for example Lactobacillusbrevis alcohol dehydrogenase. The synthesis of (+)- and (−)-α-lipoicacids starting from (+)- and (−)-8-chloro-6-hydroxyoctanoic acid alkylesters can be carried out in accordance with known methods. Theinvention is illustrated in detail by the following example.

EXAMPLE 1

100 mg (0.5 mmol) of 8-chloro-6-oxooctanoic acid methyl ester(substrate), dissolved in 50 ml of 50 mM TRIS buffer pH 7, containing0.1 mM DTT and 0.5 mM NAMP, are mixed with in each case 2 U/mg(substrate) TEADH and FDH (NADP-dependent) and stirred at 37° C. Workupby standard methods affords enantiopure (ee>99.5%)(R)-8-chloro-6-hydroxyoctanoic acid methyl ester (product).

1. A method for the production of (R)-S-chloro-6-hydroxyoctanoic acidalkyl esters of the formula (R)-II,

in which R has in each case the meaning C₁₋₄-alkyl, characterized inthat 8-chloro-6-oxooctanoic acid alkyl esters of the formula I arereduced enzymatically using alcohol dehydrogenases or carbonylreductases in the presence of a cofactor:.
 2. A method for theproduction of (S)-8-chloro-6-hydroxyoctanoic acid alkyl esters of theformula (S)-II,

in which R has in each case the meaning C₁₋₄-alkyl, characterized inthat 8-chloro-6-oxooctanoic acid alkyl esters of the formula I arereduced enzymatically using alcohol dehydrogenases or carbonylreductases in the presence of a cofactor.
 3. The method as claimed inclaim 1, characterized in that an alcohol dehydrogenase fromThermoanaerobium brokii is employed.
 4. The method as claimed in claim2, characterized in that an alcohol dehydrogenase from Lactobacillusbrevis is employed.
 5. The method as claimed in claims 1-4,characterized in that a cofactor regeneration system which shifts thereduction equilibrium is included in the enzymatic reduction.
 6. Amethod for the production of (R) or (S)-α-lipoic acid, characterized inthat an (R)- or (S)-8-chloro-6-hydroxyoctanoic acid alkyl ester obtainedby one of the methods above is converted in a manner known per se.